The two pore channels (TPCs) belong to the voltage-gated ion channel superfamily. The three TPCN genes encode proteins that are most closely related to the pore-forming subunit of voltage-gated Ca2+ and Na+ channels. Each TPC protein contains 12 putative transmembrane (TM) 1-helices with two potential pore loops. This structure feature suggests that TPCs may represent an intermediate evolutionary step from the single domain 6-TM channels to the four-domain Ca2+ and Na+ channels. Our recent studies show expression of TPC1 and TPC3 on endosomal membranes and that of TPC2 on lysosomal membranes, suggesting that TPCs are most likely Ca2+-permeable channels of intracellular organelles. We further show that TPC2 is a plausible candidate of the receptor for nicotinic acid-adenine dinucleotide phosphate (NAADP). We hypothesize that vertebrate TPCs are organelle channels involved in various aspects of intracellular vesicle trafficking, fusion, and Ca2+ signaling. The long-term objective of our research is to understand Ca2+ signaling mechanisms in mammalian cells. It has long been recognized that NAADP releases Ca2+ from an internal pool that is distinct from the endoplasmic reticulum. More recent studies show that the NAADP-sensitive pool is associated with lysosome-like acidic compartments. Thus, the study of TPC2 as an NAADP receptor provides a unique opportunity to address this rather distinct, but also poorly understood, pathway of Ca2+ signaling. The goals of the proposed research are to test the aforementioned hypothesis and to set a foundation for a new area of Ca2+ signaling focusing on the structures and functions of organelle Ca2+ permeable channels. We have three specific aims. Aim 1 will test the hypothesis that TPC2 is an NAADP receptor. We will examine the binding of NAADP to TPC2-containing membranes and study NAADP-evoked Ca2+ response in cells overexpressing TPC2 as well as those that have TPC2 expression knocked down by RNA interference. Aim 2 will examine a novel intracellular mechanism by which membrane stretch stimulates NAADP production, causing Ca2+ release from lysosomal stores via the TPC2 channel. Aim 3 will examine the molecular dynamics of TPC1-3 in subcellular vesicle trafficking and to determine sorting signals for the organelle targeting of TPCs. These comprehensive studies will greatly enhance our understanding on these novel ion channels and shed light on their potential roles in normal human physiology and pathophysiology. PUBLIC HEALTH RELEVANCE: Two pore channels (TPCs) are novel ion channels related to voltage-gated calcium channels but they are expressed on intracellular organelles such as endosomes and lysosomes instead of plasma membrane. One of them, TPC2, is likely involved in mediating calcium release from lysosomes in response to the second messenger, nicotinic acid-adenine dinucleotide phosphate (NAADP). The proposed project will elucidate the molecular functions of TPCs in various aspects of intracellular vesicle trafficking, fusion, and calcium signaling and shed light on their potential roles in normal human physiology and pathophysiology.